DNA binding activity of vesicles produced by competence deficient mutants of Haemophilus

Kahn, Marc E.; Concino, Michael F.; Gromkova, Rosa; Goodgal, Sol H.

doi:10.1016/0006-291x(79)92024-2

Cited by 26 publications

(14 citation statements)

References 7 publications

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“…b-MVs were incubated with DNA at the ratio at which these occurred within the matrix. In agreement with the DNA-retaining properties of p-MVs (11,12,21,39), b-MVs showed a substantial increase in associated DNA (281 Ϯ Ͻ1 ng DNA/20 g MV protein [mean Ϯ SEM; n ϭ 60]). DNase-treated MVs showed a propensity similar to that for native MVs (269 Ϯ 1 ng DNA/20 g MV protein [mean Ϯ SEM; n ϭ 60]), suggesting that, for a given amount of DNA, there is a maximum DNA load per unit MV protein.…”

Section: Resultssupporting

confidence: 70%

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Interactions of DNA with Biofilm-Derived Membrane Vesicles

2009

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The biofilm matrix contributes to the chemistry, structure, and function of biofilms. Biofilm-derived membrane vesicles (MVs) and DNA, both matrix components, demonstrated concentration-, pH-, and cationdependent interactions. Furthermore, MV-DNA association influenced MV surface properties. This bears consequences for the reactivity and availability for interaction of matrix polymers and other constituents.The biofilm matrix contributes to the chemistry, structure, and function of biofilms and is crucial for the development of fundamental biofilm properties (46,47). Early studies defined polysaccharides as the matrix component, but proteins, lipids, and nucleic acids are all now acknowledged as important contributors (7, 15). Indeed, DNA has emerged as a vital participant, fulfilling structural and functional roles (1,5,6,19,31,34,36,41,43,44). The phosphodiester bond of DNA renders this polyanionic at a physiological pH, undoubtedly contributing to interactions with cations, humic substances, fine-dispersed minerals, and matrix entities (25,41,49).In addition to particulates such as flagella and pili, membrane vesicles (MVs) are also found within the matrices of gram-negative and mixed biofilms (3,16,40). MVs are multifunctional bilayered structures that bleb from the outer membranes of gram-negative bacteria (reviewed in references 4, 24, 27, 28, and 30) and are chemically heterogeneous, combining the known chemistries of the biofilm matrix. Examination of biofilm samples by transmission electron microscopy (TEM) has suggested that matrix material interacts with MVs ( Fig. 1). Since MVs produced in planktonic culture have associated DNA (11,12,13,20,21,30,39,48), could biofilm-derived MVs incorporate DNA (1, 39, 40, 44)? MATERIALS AND METHODSBiofilm growth and isolation and purification of matrix and MVs. Pseudomonas aeruginosa PAO1 and green fluorescent protein-tagged PAO1 (17) biofilms were grown using the agar plate model (40). Matrix was isolated (40) and sequentially filtered through 0.22-, 0.45-, and 1.2-m cellulose acetate filters and the filtrate collected. Absence of cells was confirmed by plating 100-l aliquots (18 h at 37°C; trypticase soy agar; n ϭ 3) and TEM of whole-mount preparations (see below). Matrix for characterization was dialyzed (24 h at 4°C; Spectrapor regenerated cellulose dialysis membranes [molecular mass cutoff, 3,000 Da]; Fisher). Particulate components were harvested by ultracentrifugation (125,000 ϫ g for 1.5 h at 5°C; Beckman Ti45 rotor), the pellets resuspended in 30% (vol/vol) Optiprep (Sigma), and the components separated on Optiprep density gradients (2) at 0% (1 ml), 18% (1 ml), 20% (1 ml), 22.5% (3 ml), 25% (3 ml), 27.5% (3 ml), 30% (3 ml), and 50% (1 ml) (vol/vol). All Optiprep solutions contained 10 mM HEPES, 0.85% (wt/vol) NaCl (pH 7.4). Samples were centrifuged to equilibrium (100,000 ϫ g for 16 h at 5°C; Beckman SW28.1 rotor) and fractionated (200-l aliquots) and whole mounts assessed by TEM (see below). MV-containing fractions were combined, washed twice in water (1...

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Section: Resultssupporting

confidence: 70%

“…1). Since MVs produced in planktonic culture have associated DNA (11,12,13,20,21,30,39,48), could biofilm-derived MVs incorporate DNA (1, 39, 40, 44)? …”

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confidence: 99%

Interactions of DNA with Biofilm-Derived Membrane Vesicles

2009

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“…The type b capsule of H. injluenzae is reported to consist of a polymer of ribosylribitol phosphate (Zamenhoff et al, 1953;Crisel et al, 1975). Recent studies on cellular changes that occur during the development of competence have provided evidence that competence is associated with the appearance of vesicles on the outer membrane (Kahn et al, 1979;Kahn & Smith, 1984). These vesicles (called 'transformasomes') have been implicated in the uptake and transport of DNA during transformation (Kahn et al, 1983).…”

Section: Discussionmentioning

confidence: 99%

Genetic Transformation in Encapsulated Clinical Isolates of Haemophilus influenzae Type b

Rowji

Gromkova²,

Koornhof³

1989

Microbiology

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Haemophilus inzuenzae type b strains isolated from children with meningitis, septicaemia and pharyngitis were studied for their ability to undergo genetic transformation by two chromosomal markers, streptomycin resistance and nalidixic acid resistance. Fifty-eight percent of the strains were non-transformable while the remaining 42% showed considerable strain variation with regard to their transformation frequencies, which ranged from 8 x to 1 xThe effect of type b capsule on competence development and transformation activity was studied by comparing encapsulated strains with their non-encapsulated variants. Type b capsule did not inhibit either competence development or transforming efficiency. The lack of transformability in the majority of strains was not due to the presence of a capsule.

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“…[2][3][4][5]. Although various changes in outer and inner membrane composition have been observed during competence induction (6)(7)(8)(9), little is known about the actual biochemistry and mechanics of DNA uptake. Furthermore, it is puzzling how donor DNA, which remains duplex after uptake, escapes restriction and degradation by cellular nucleases while awaiting integration.…”

mentioning

confidence: 99%

“…First, membranous extensions are internalized as a consequence of the addition of homologous donor DNA to competent H. parainfluenzae cells, and membrane-donor DNA complexes have been isolated from mechanically disrupted cells (10). Second, studies with competence-deficient vesicle-shedding mutants of both H. influenzae and H. parainfluenzae strongly suggest that the DNA binding activity associated with competent cultures resides exclusively on membranous extensions (9,12). From these observations, it has been proposed that during binding and uptake, donor DNA is in some way packaged into this unique membrane body, which we have renamed the "transformasome.…”

mentioning

confidence: 99%

Transformasomes: specialized membranous structures that protect DNA during Haemophilus transformation.

Kahn¹,

Barany²,

Smith³

1983

Proc. Natl. Acad. Sci. U.S.A.

113

100

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The mechanism by which Haemophilus protects donor DNA from cellular restriction and degradative enzymes during transformation is unclear. In this report, we demonstrate that donor DNA enters Haemophilus influenzae through specialized membranous extensions, which we have termed "transformasomes." DNA within transformasomes is in a protected stateresistant to external DNase and cellular restriction enzymes, although remaining unmodified and double-stranded. The ability of donor DNA to exit from transformasomes is dependent on its topological conformation. Circular DNA remains intact within transformasomes, while linear DNA rapidly exits and undergoes homologous recombination. Protected donor DNA can be preferentially removed from the surface of competent cells by extraction with organic solvents. Structurally intact transformasomes containing donor DNA could be partitioned into the organic layer and can be further purified by density centrifugation.Haemophilus influenzae is a Gram-negative bacterium that can be induced to high levels of competence for transformation under conditions of slowed growth (1). Competent cells efficiently take up homologous donor DNA from the medium and integrate it into the chromosome to yield transformation rates of 1-3% for various genetic markers (for reviews, see refs. 2-5). Although various changes in outer and inner membrane composition have been observed during competence induction (6-9), little is known about the actual biochemistry and mechanics of DNA uptake. Furthermore, it is puzzling how donor DNA, which remains duplex after uptake, escapes restriction and degradation by cellular nucleases while awaiting integration. Recently, Kahn et al. (10) have suggested that specialized membranous extensions on the surface of competent cells might be responsible for capturing and protecting the donor DNA after uptake. By comparative morphological studies, these membranous structures were shown to appear on the surface of H. influenzae and Haemophilus parainfluenzae as they become competent (10, 11). The structures extend "35 nm from the cell, average 20 nm in diameter, are located at points of fusion between the inner and outer membrane, and are composed of a lipid bilayer-predominantly outer membrane proteins (10, 12) and lipopolysaccharide. Pore structures with an opening of 5 nm are localized at points of attachment of the membranous extension to the outer membrane. (An electron micrograph of this structure is seen in Fig. 5 KW22 and KW23 (13), were grown in 2.5% heart infusion broth (Difco), supplemented with 10 ,4g of hemin (Eastman) and 2 ,ug of NAD (Sigma) per ml. Competent cells, routinely able to transform at 3% for chromosomal markers, were prepared in MIV media as described (1). Plasmids pPUP3 (pBR322 containing an 11-basepair uptake site in the Pst I site) (14) and pCML6 were isolated from Escherichia coli strains HB101 (r-, m-, recA) and MM294 (endA hsdR) by the procedure of Birnboim and Doly (15)

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DNA binding activity of vesicles produced by competence deficient mutants of Haemophilus

Cited by 26 publications

References 7 publications

Interactions of DNA with Biofilm-Derived Membrane Vesicles

Interactions of DNA with Biofilm-Derived Membrane Vesicles

Genetic Transformation in Encapsulated Clinical Isolates of Haemophilus influenzae Type b

Transformasomes: specialized membranous structures that protect DNA during Haemophilus transformation.

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